Studies on production and purification of protease from Bacillus megaterium au02

Abstract

Proteases are enzymes which catalyze the hydrolysis of peptide newlinebonds in proteins and peptides Microbial proteases are currently receiving newlinemuch attention because of their potential use in a variety of industries newlineBiocatalysis in organic solvents has emerged as an area of systematic research newlineand industrial development fueled mainly by the interest of chemical and newlinepharmaceutical majors Solvent stability remains a pre requisite for newlineemploying enzymes in a non aqueous system Enzymes from solvent tolerant newlinemicrobes appear to be the catalysts of choice for developing solvent stable newlineenzymes The first objective of the present work is the isolation and newlineidentification of solvent tolerant bacterium that produces extracellular solvent newlinestable protease The second objective of the work is to optimize culture newlineconditions and production media by statistical design methods to improve newlinesecretion of extracellular protease The third objective is to purify the newlineextracellular protease to homogeneity The fourth objective of the work is the newlinebiochemical characterization of the organic solvent stable protease newlineA solvent tolerant strain AU02 producing extracellular protease newlinewas isolated from milk processing industrial wastes and was identified as newlineBacillus megaterium by 16S rDNA sequencing The process parameters newlineinfluencing the production of extracellular protease were identified by onefactor newlineat a time approach followed by Plackett Burman design and further newlineiv optimized by Central Composite Design CCD and Response Surface newlineMethodology RSM Eight variables fructose skim milk CaCl2 MgSO4 newlinetemperature initial pH of medium inoculum size and incubation time newlineaffecting protease production were identified by one factor at a time newlineapproach their effects were evaluated using Plackett Burman design RSM newlinewas applied for most significant parameters skim milk CaCl2 inoculum size newlineand incubation time to determine maximum protease production The newlineexperimental result 43 0 U ml in a medium optimized for protease newlineproduction was in good agreement with the predicted value of a quadratic newlinemodel 459 Uml thus confirming its validity newline newline